Electrophoretic separation techniques are based upon the differential mobilities of the components of a mobile phase passing through a stationary separation medium under the influence of an applied electric field. The components are distinguished by their migration times past a fixed point in the electrophoretic pathway or by their positions within the pathway at a fixed time. Capillary electrophoresis (CE) is one example of the former while capillary isoelectric focusing (IEF) is an example of the latter. The separation medium in free solution CE is the buffer filled capillary tube itself.
Instruments for performing capillary electrophoresis are frequently designed as flow-through systems. In IEF the separated components are commonly mobilized past a fixed detector following separation. The capillaries must also be washed between sample runs. In CE complicated hydraulic systems are required to accurately control sample introduction. A component's velocity is the vector sum of the bulk flow velocity, due to electroosmotic force, and the component's electrophoretic velocity. In capillary IEF, tubes are typically coated on their interior surface to eliminate electroosmosis and buffer reservoirs of high pH at the anode and low pH at the cathode are located at either end of the capillary tube. Components are focused within a stationary pH gradient to their isoelectric points and then mobilized by a variety of methods past a detector. CE capillaries vary in length from 70 mm to 1000 mm, with longer lengths used to improve resolution at the price of increased run time. IEF capillaries are typically 20-100 mm in length. Flow-through systems typically employ one capillary and can only separate one sample at a time. Additional controls, calibrators, and samples must be run sequentially. The use of long capillaries in CE, and the requirement for mobilization past a detector in IEF, greatly increase analysis time per sample.
Automated systems designed to perform multiple runs on different samples require wash cycles between runs. This significantly increases the volume of liquid waste produced. Reservoirs are required for wash solutions, waste and reagent which must be monitored and serviced by trained personnel. If the sample contains biohazardous material then waste disposal and instrument contamination become additional problems. Cross contamination resulting from electrode contamination is a particular problem. Auto-samplers which quantitatively load a sample into the system are typically designed to operate sequentially on samples and generally incorporate a wash cycle between samples.
Cartridges containing a capillary tube for insertion into a capillary electrophoresis instrument are known. U.S. Pat. No. 4,985,129 to Burd discloses a planar cartridge containing a looped capillary and a pair of aligned windows between which a segment of the capillary tube passes for zone detection. The cartridge is designed to connect the capillary tube ends to external reservoirs. U.S. Pat. No. 5,037,523 to Weinberger et al. discloses a similar cartridge further including air cooling slots in the cartridge body and annular electrodes surrounding the capillary tube ends. The cartridge is also designed to connect the capillary tube ends to external reservoirs.
U.S. Pat. No. 4,816,123 to Ogan et al. discloses a method for forming capillary electrophoresis channels using a wire or capillary tube as a template strand. Detectors are positioned next to the template strand prior to molding a plastic material around the template strand and detectors.
U.S. Pat. No. 4,908,112 to Pace discloses a capillary sized conduit constructed by covering an etched channel in a silicon wafer with a glass plate. Reservoirs are located at either end of the channel which is intersected by a second channel used for sample introduction. Electrodes are located throughout the system so that liquids may be moved by electroosmosis. Multiple channels, which are filled with a gel preparation fluid by capillary action from overhead reservoirs containing electrodes, are also disclosed. The reservoirs are then filled with buffer and a sample is injected into the reservoir with a volumetric syringe. The electroosmotic channels are less than 100 .mu.m in cross-sectional dimension while the gel-filled channels are greater than 100 .mu.m in cross-sectional dimension. None of the prior art devices contain only those portions of the electrophoretic separation system which contact the sample.
Devices for sample loading in capillary electrophoresis are known. U.S. Pat. No. 4,911,807 to Burd discloses a cassette having short capillary segments which are sequentially introduced into an electrophoretic pathway for sample loading or fraction collecting. U.S. Pat. Nos. 4,906,344 and 5,073,239 to Hjerten disclose thermal and electroendosmotic pumping means respectively for quantitative sample injection in capillaries. Mechanical pumps are also frequently employed. All of these devices require external manipulation of the system by some means to produce a quantitative sample load.
Use of one or more absorbent materials to provide motive force to fluid samples in disposable assay devices is disclosed in U.S. Pat. Nos. 5,006,309 to Khalil et al. and 5,006,474 to Horstman et al. Khalil et al. disclose an immunoassay device in which an absorbent material pulls fluid through an immobilizing fiber matrix where the results of the assay can be read. Horstman et al. disclose a device where two absorbent materials cause lateral bi-directional flow through a chromatographic separation material. Neither device uses a differential rate of flow to quantitatively load a sample into a fixed volume.
Most capillary electrophoresis instruments are flow-through devices which generate large volumes of waste relative to the effective separation volume of the capillaries. A significant portion of this waste volume arises from the need to wash those portions of the system which come in contact with the sample. These include the sample loaders, electrodes, buffer reservoirs and capillaries. These devices are generally incapable of running simultaneous multiple lane separations.
An object of the invention is to provide a single use separation cartridge containing all of those portions of an electrophoretic separation system which contact the sample.
Another object of the invention is to provide a single use separation cartridge which is capable of automatic quantitative sample loading.
A further object of the invention is to provide a single use separation cartridge that uses capillary forces to quantitatively introduce sample and buffers into the capillary.
A further object of the invention is to provide a single use separation cartridge that replaces large sample reservoirs with hemispherical drops of sample and buffer.
A further object of the invention is to provide a single use separation cartridge that contains all necessary reagents, wash solutions and waste receptacles.